Mycobacterial infections result in the activation of macrophage signaling pathways that are essential for stimulating a host immune response. However our understanding is limited as to which signaling pathways are transduced in macrophages upon mycobacterial invasion and whether pathogenic mycobacteria modulate these signals. In our experiments to define the macrophage signaling pathways initiated during a mycobacterial infection we determined that the mitogen activated protein kinases (MAPK) p38 and ERK 1/2 were activated in murine macrophages upon mycobacterial infection. However, the MAPK activity was limited in macrophages infected with pathogenic M. avium strains relative to cells infected with non-pathogenic mycobacteria. A limited production of TNF-alpha was also observed in these M. avium infected cells. Inhibitor studies indicated that the MAPKs were required for the mycobacteria-mediated induction of TNF-alpha. Moreover, macrophages infected with a glycopeptidolipid (GPL) deficient M. avium 2151 also showed increased MAPK activation and TNF-alpha production compared to cells infected with a isogenic 2151 strain containing GPLs. Therefore, we hypothesize that the MAPKs are key components in the macrophage signaling pathways initiated during a mycobacterial infection and that M. avium has evolved mechanisms to limit their activity in part through production of GPLs. Additional analysis showed that modifying the GPL structure through deletion of the methyl transferase D gene resulted in a M. avium 104 strain with attenuated virulence. Thus, we propose the following specific aims: 1) produce a panel of M. avium 724 and 104 mutants that lack specific genes involved in GPL biosynthesis 2) Biochemically characterize the M. avium mutants for GPL structure and cell wall composition 3) Characterize GPLs for their affect on macrophage activation and mycobacterial virulence. Our long-term goal is to better understand the macrophage signaling pathways initiated during a mycobacterial invasion. We believe a more careful analysis of these macrophage responses and how mycobacteria may modify them will lead to novel insights into mycobacterial pathogenesis.